3. Electronic Theses and Dissertations (ETDs) - All submissions
Permanent URI for this communityhttps://wiredspace.wits.ac.za/handle/10539/45
Browse
4 results
Search Results
Item An investigation into some of the organic constituents of soft and hard tissues of the body(1957) Solomons, C.C.The roles played by fibrous proteins in nature are at present being intensively studied and some of the advances in our knowledge of these proteins are the subject of several recent symposia. (la,b) The powerful techniques of X-ray diffraction end electron— optic analysis as well as ran unprecendented improvement in the specificity and accuracy of chemical and chromatographic methods of analysis has led to ever increasing efforts to interpret biological phenomena within a framework of physicochemical principles. In many fields, particularly those of virus research and muscle contraction these efforts have already been rewarded by a large measure of success. In the present work, which forms part of a program of research into the chemistry of connective tissues of man and other vertebrates, the chemical properties and structural features of the extracellular collagenous proteins in some calcified and uncalcified connective tissues are compared. 'In vivo' calcification of connective tissue is generally thought to be the result of a physico-chemical combination between lima salt3 and the organic matrix mediated by the ionic and structural prop rties of the matrix and the activity of various cello and enzymesItem Class pi glutathione S-transferase: unfolding and conformational stability in the absence and presence of G-site ligands(1996) Erhardt, JulijaThe glutathione S-transferases (GST) are a supergene family of h0111o-or heterodimeric Phase II detoxification enzymes which catalyse the S-conjugation between glutathione and an electrophilic substrate. The active site can be divided into two adjacent functional regions; a highly specific Gssite for binding the physiological substrate glutathione and a nonspecific If-site for binding nonpolar electrophilic substrates. Unfolding of porcine class Pi isoenzyme (pGSTPl~l) was monitored under equilibrium conditions using different physicochemical parameters. The coincidence of unfolding curves obtained with functional and structural probes, the absence of thermodynamically stable intermediates such as a folded monomer, and the dependence of pGSTPl··l stability upon protein concentration, indicate a cooperative and concerted two-state unfolding transition between native dimeric pGSTPl-l and unfolded monomeric enzyme. Equilibrium and kinetic unfolding experiments employing tryptophan fluorescence and enzyme activity measurements were preformed to study the effect of ligand binding to the G-site on the unfolding and stability of the porcine class pi glutathione S-transferase against urea. The presence of glutathione caused a shift in the equilibrium-unfolding curves towards lower urea concentrations and enhanced the first-order rate constant for unfolding suggesting a destabilisation of the pGSTPl-l structure against urea. The presence of either glutathione sulphonate or S-hexylglutathione, however, produced the opposite effect in that their binding to the G-site appeared to exert a stabilising effect against urea. The binding of these glutathione analogues also reduced significantly the degree of cooperativity of unfolding indicating a possible change in the protein's unfolding pathway.Item The role of electrostatic interactions in the stability and structural integrity of human CLIC1(2012-02-23) Legg-E'Silva, Derryn AudreyChloride intracellular channel proteins (CLICs) are able to exist in a soluble or membrane-bound state. The mechanism by which the transition between the two states takes place is yet to be elucidated. It is proposed that structural rearrangements of the N-terminal domain take place when CLICs encounter the lower pH environment of the membrane surface (pH 5.5). This prompts the CLICs to form a soluble membrane-ready state prior to pore formation and membrane transversion. Since the insertion of CLIC1 into membranes occurs at low pH, perhaps protonation and electrostatic effects of key conserved residues at the domain interface situated within the transmembrane region bring about the structural changes necessary for this transition. Structural and sequence alignments revealed that a conserved salt-bridge interaction between conserved residues on helices 1 and 3 of the N-terminal domain is present at the domain interface of CLICs. Therefore, this interaction was proposed to play an important role in maintaining the structural integrity and conformational stability of the N-terminal domain. This hypothesis was tested by mutating conserved CLIC1 residues Arg29 and salt-bridge partner Glu81 to methionine, thus removing the salt-bridge interaction. The conformational stabilities of each mutant at pH 7 (cytosol) and pH 5.5 (membrane surface) in the absence of membranes was then measured and compared to that of the wild type protein. The mutations did not impact upon the structural integrity of the protein. However, removal of the salt-bridge and hydrogen bonding interactions caused a loss in the cooperativity of unfolding from the native to unfolded state that resulted in the formation of an intermediate species. The intermediate species are less stable than the intermediate species of wild type CLIC1 at pH 5.5. Nevertheless, the properties (secondary and tertiary structure, ANS binding and cooperative unfolding (N ↔ U)) of the intermediate species are the same for all mutants and wild type protein. It can be concluded that the salt-bridge and more importantly hydrogen bonding interactions between helices 1 and 3 stabilise the Nterminal domain of CLIC1. It can be hypothesised that in the absence of membranes under acidic conditions, such as those at the surface of the membrane, protonation of acidic amino acid residues at the domain interface cause destabilisation of the Nterminal domain. This causes a reduction in the activation energy barrier for the conversion of soluble CLIC1 to its membrane-insertion conformation.Item The role of the domain interface in the stability, folding and function of CLIC1(2008-09-08T08:36:18Z) Stoychev, Stoyan HristovChloride intracellular channel protein 1 (CLIC1) is a dual-state protein existing in both soluble monomeric conformation as well as integral-membrane form. The role of the domain interface in the conversion between these species was investigated. Bioinformatics-based analysis was undertaken to compare and contrast the domain interfaces of dimeric GSTs with their monomeric homologues CLIC1 and CLIC4. The mutants CLIC1-M32A and CLIC1-E81M were used as experimental case studies on the role of domain-domain interactions in the stability and folding of CLIC family proteins. A consensus interface was revealed with the prominent interaction being a conserved inter-domain lock-and-key type motif previously studied in class Alpha GSTs (Wallace et al., 2000). A number of domain-interface interactions were found to be unique to the CLIC family and as such thought to play a role in the conversion of these proteins from their soluble form to an integral membrane form. Overall the domain interfaces of monomeric CLIC1 and CLIC4 did not differ significantly from the domain interfaces of dimeric GSTs. The removal of the unique CLIC family salt-bridges between Arg29 and Glu81 and the cavity forming domain interface mutation Met32Ala did not induce significant changes in the conformational flexibility of the native state. The true role of the Arg29-Glu81 salt-bridges was masked by the introduction of stabilizing hydrophobic contacts. Removal of the inter-domain lock-and-key interaction destabilized CLIC1 significantly with concomitant loss in cooperative folding that resulted in the stabilization of a molten globule-like species. This intermediate state was less stable and less structured than the equilibrium intermediate of wtCLIC1 at pH 5.5. However the bulk of the structures found to unfold during intermediate-species formation was the same in mutant and wild-type proteins. It was concluded that formation of the membrane-competent form of CLIC1 involves re-structuring of the N-terminal thioredoxin domain that takes place after destabilization of the salt bridges connecting h1 and h3 and uncoupling of the inter-domain lock-and-key motif.